Compositions and methods for tumor transduction

ABSTRACT

The invention relates to cancer therapeutics, in particular, the system of making cancer cells more susceptible to effector cells by introduction of cellular therapy targets into the cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/249,183 filed Oct. 30, 2015, the entire contents of which ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

Recent examples of chimeric antigen receptor (CAR) T cell directed tosolid tumors have been clinical (or even preclinical) failures. Theinvention described herein presents solutions for solid tumor and otheroncology indications.

Cellular therapeutics that are introduced into cancer patients andtraffic into tumor microenvironments encounter multiple barriers thatlimit their efficacy. These barriers include, but are not limited to:cellular energy, intrinsic cell death, suboptimal cell trafficking,limited proliferation, limited effector function, poor “take” (survival,persistence and differentiation), active exclusion from tumors,persistent immunosuppression in the tumor microenvironment, andtumor-induced cell death. Almost all attempts to date to overcome theselimitations require non-physiological manipulation of the cellulartherapeutic both in vitro and in vivo. The efficacy of cellulartherapeutics is therefore broadly hindered by both intrinsic andextrinsic factors.

Targeting solid tumors presents an additional set of challenges toovercome, for example, their overall lesser sensitivity to T cellmediated cytotoxicity, a microenvironment with differingimmunosuppressive mechanisms between tumor types, and a lack of targetantigens with favorable expression profiles. Additionally, solid tumorsare heavily fortified against cellular therapeutic attack as they deployan impenetrable extracellular matrix, hypoxia, and acidic pH. Despitethe vast number of targets that have been investigated, only a smallnumber are tumor-specific (e.g., expression is restricted to the tumorcell) and this finding, as such, illustrates the difficulty and the needto discover an effective solution for eliminating or reducing the tumor.Yet another problem for targeting tumors is the heterogeneity of thetumor cells which express different antigens and different levels ofantigens. The invention described herein addresses these problems andprovides additional benefits as well.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, upon the introductionof cellular therapy targets into cancer cells (e.g., tumor cells such assolid tumor cells) such that they are more susceptible to effector cellsthat can eliminate and/or reduce the cancer cells. As further detailedherein, cancer cells (e.g., tumors) can be transduced with fusionproteins. Such fusion proteins consist of a binding component fused to atarget component. For example, the fusion protein can be an antibody orantibody fragment which binds to one or more tumor-associated antigens(TAA) or tumor-specific antigens (TSA), fused to a target for cellulartherapy, e.g. CD19; a cytokine-target fusion; a bi-specific T-cellengager (BiTE). They can also be transduced with CD19 variants (ormutants). These fusion proteins or variants (or mutants) can be secretedby the tumor cells and/or permeate the tumor microenvironment.

Accordingly, in one aspect, the invention provides for recombinantvectors encoding an antibody or antibody fragment to a tumor-associatedantigens (TAA) or tumor-specific antigens (TSA) and a therapeutic agent,as a fusion protein. In other embodiments, the therapeutic agent isselected from the group consisting of a cytokines, peptides, proteins,antibodies, antibody fragments, T-cell engager and NK-cell engager. Inother embodiments, the therapeutic agent is selected from the groupconsisting of glioma-associated antigen, carcinoembryonic antigen (CEA),β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactiveAFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reversetranscriptase, RU1, RU2 (AS), intestinal carboxyi esterase, mut hsp70-2,M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1,LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin and telomerase,prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophilelastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-Ireceptor and mesothelin, EphA2, HER2, GD2, Glypican-3, 5T4, 8H9, αvβ6integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa lightchain, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138,CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4,ErbB3/4, FAP, FAR, FBP, fetal AchR, Folate Receptor a, GD2, GD3, HLA-AIMAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP,Mesothelin, Mucl, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA,PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonicantigen, HMW-MAA, and VEGF receptors.

In other embodiments, the cytokine is capable of being secreted by atumor cell comprising said vector. In other embodiments, the vector is aviral vector. In other embodiments, the vector integrates into thegenome of a cancer cell. In other embodiments, the therapeutic agent isan antigen is an antigen for a chimeric antigen receptor (CAR). In otherembodiments, the therapeutic agent is an antigen for a T cell receptor(TCR). In other embodiments, the therapeutic agent is an antigen for anADC antibody, ADCC antibody, and/or a radiotherapeutic antibody. Inother embodiments, the therapeutic agent is an immunostimulatorycytokine or molecule selected from the group consisting of TLR agonists,PAMP, DAMP and other stimulators. In other embodiments, the therapeuticagent is a peptide with immunomodulatory or anti-tumor properties. Inother embodiments, the TAA or TSA and therapeutic agent are expressed asa fusion protein. In other embodiments, the TAA or TSA is selected fromthe group consisting of a glioma-associated antigen, carcinoembryonicantigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP),lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerasereverse transcriptase, RU1, RU2 (AS), intestinal carboxyi esterase, muthsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP,NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2/neu, survivin andtelomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M,neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I,IGF-II, IGF-I receptor and mesothelin, EphA2, HER2, GD2, Glypican-3,5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CA9, CD19, CD20,CD22, kappa light chain, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70,CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM,ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR, Folate Receptor a,GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y,MCSP, Mesothelin, Mud, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME,PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2,carcinoembryonic antigen, HMW-MAA, and VEGF receptors

In other aspects, the invention provides for recombinant vectorsencoding a cytokine to a tumor expressed cytokine receptor and atherapeutic agent as a fusion protein. In some embodiments, the cytokinereceptor is a type I receptor or a type II receptor.

In other aspects, the invention provides for recombinant tumor cellscomprising a vector as described above and herein. In some embodiments,the tumor cell expresses one or more fusion proteins.

In other aspects, the invention provides for methods of producing arecombinant tumor cell capable of expressing a fusion protein comprisingan antibody or antibody fragment to a tumor-associated antigens (TAA) ortumor-specific antigens (TSA) and a therapeutic agent, said methodcomprising (a) introducing a vector as described above and herein into atumor cell; and (b) culturing the tumor cell such that the vector istransduced into the tumor cell. In some embodiments, the vector and/orits components integrate in the tumor cells' genome. In someembodiments, the TAA or TSA is selected from the group consisting ofglioma-associated antigen, carcinoembryonic antigen (CEA), β-humanchorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP,thyroglubilin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase,RU1, RU2 (AS), intestinal carboxyiesterase, mut hsp70-2, M-CSF,prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53,prostein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinomatumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2,CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor andmesothelin, EphA2, HER2, GD2, Glypican-3, 5T4, 8H9, αv⊖6 integrin, BCMA,B7-H3, B7-H6, CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30,CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA,CSPG4, EGFR, EGFRvIII, EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP,FAR, FBP, fetal AchR, Folate Receptor a, GD2, GD3, HLA-AI MAGE A1,HLA-A2, IL11Ra, IL13Ra2, KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Mucl,Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1,SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, carcinoembryonic antigen, HMW-MAA,and VEGF receptors. In some embodiments, the TAA or TSA is an antigenfor a chimeric antigen receptor (CAR). In some embodiments, the TAA orTSA is an antigen for a T cell receptor (TCR). In some embodiments, thetherapeutic agent is an antigen for an ADC antibody, ADCC antibody,and/or a radiotherapeutic antibody. In some embodiments, the therapeuticagent is an immunostimulatory cytokine or molecule selected from thegroup consisting of TLR agonists, PAMP, DAMP and other stimulators. Insome embodiments, the therapeutic agent is a peptide withimmunomodulatory or anti-tumor properties.

In other aspects, the invention provides for methods of treating cancerin an individual in need thereof comprising administering a compositioncomprising a vector as described above and herein. In some embodiments,a fusion protein is expressed. In other embodiments, the cancer cellexpresses a therapeutic agent that increases immune response against thecancer cell. In some embodiments, the cancer cell express one or moreprotein or peptide antigens that is capable of being recognized by aCAR. In some embodiments, the cancer cell express one or more protein orpeptide antigens that is capable of being recognized by a TCR. In someembodiments, the cancer cell express one or more fusion proteinscontaining therapeutic agents capable of being recognized by immunecells in the individual. In some embodiments, the method furthercomprises administration of CAR T cells to the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing a non-limiting example of the methods ofintroduction of a cellular therapy target into solid tumor cells. Stepone corresponds to the transduction construct (e.g., tumor-selectivetransduction via e.g., viral transduction, RNA transduction, and geneinsertion), and/or in vivo transduction and integration. Step twocorresponds to the expression of a fusion protein (e.g., an antibodyfragment to a tumor antigen fused to a CAR antigen, for example an scFvanti-tumor associated antigen (TAA) fused to CD19 or a fragmentthereof). Step three corresponds to the antibody fragment fused to anantigen, e.g. CD19, which binds to tumor cells and presents the antigen,and all nearby tumor cells are coated with the antibody fragment fusedto a protein, peptide, or other agent, for example, CD19, which is anantigen recognized by CAR T cells. Step four corresponds to the infusionin vivo of CAR T cells that recognize the CAR antigen (e.g., anti-CD19CAR T cells). The response becomes cytotoxic to the CD19-coated tumorcell.

FIGS. 2A-2C demonstrate expression of fusion proteins from AAVtransduced cells.

FIGS. 3A and 3B show summary results of an IFNγ ELISA measuringinduction of IFNγ in CAR19 T-cells by expressed fusion proteins.

FIGS. 4A-4C show induction of cytotoxicity upon incubation of CAR19 Tcells with AAV-1-expressed supernatant and BT474 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, upon the introductionof cellular therapy targets into solid tumor cells. Tumor cells can betransduced with fusion proteins such that the fusion proteins aresecreted by the tumor cells and released to the tumor microenvironmentto combat the tumor.

I. Definitions

As used herein, the term “antibody” refers to a polypeptide thatincludes canonical immunoglobulin sequence elements sufficient to conferspecific binding to a particular target antigen. As is known in the art,intact antibodies as produced in nature are approximately 150 kDtetrameric agents comprised of two identical heavy chain polypeptides(about 50 kD each) and two identical light chain polypeptides (about 25kD each) that associate with each other into what is commonly referredto as a “Y-shaped” structure. Each heavy chain is comprised of at leastfour domains (each about 110 amino acids long)—an amino-terminalvariable (VH) domain (located at the tips of the Y structure), followedby three constant domains: CH1, CH2, and the carboxy-terminal CH3(located at the base of the Y's stem). A short region, known as the“switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the antibody. Twodisulfide bonds in this hinge region connect the two heavy chainpolypeptides to one another in an intact antibody. Each light chain iscomprised of two domains—an amino-terminal variable (VL) domain,followed by a carboxy-terminal constant (CL) domain, separated from oneanother by another “switch”. Intact antibody tetramers are composed oftwo heavy chain-light chain dimers in which the heavy and light chainsare linked to one another by a single disulfide bond; two otherdisulfide bonds connect the heavy chain hinge regions to one another, sothat the dimers are connected to one another and the tetramer is formed.Naturally-produced antibodies are also glycosylated, typically on theCH2 domain. Each domain in a natural antibody has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1,FR2, FR3, and FR4). When natural antibodies fold, the FR regions formthe beta sheets that provide the structural framework for the domains,and the CDR loop regions from both the heavy and light chains arebrought together in three-dimensional space so that they create a singlehypervariable antigen binding site located at the tip of the Ystructure. The Fc region of naturally-occurring antibodies binds toelements of the complement system, and also to receptors on effectorcells, including for example effector cells that mediate cytotoxicity.As is known in the art, affinity and/or other binding attributes of Fcregions for Fc receptors can be modulated through glycosylation or othermodification. In some embodiments, antibodies produced and/or utilizedin accordance with the present disclosure include glycosylated Fcdomains, including Fc domains with modified or engineered suchglycosylation. For purposes of the present disclosure, in certainembodiments, any polypeptide or complex of polypeptides that includessufficient immunoglobulin domain sequences as found in naturalantibodies can be referred to and/or used as an “antibody”, whether suchpolypeptide is naturally produced (e.g., generated by an organismreacting to an antigen), or produced by recombinant engineering,chemical synthesis, or other artificial system or methodology. In someembodiments, an antibody is polyclonal; in some embodiments, an antibodyis monoclonal. In some embodiments, an antibody has constant regionsequences that are characteristic of mouse, rabbit, primate, or humanantibodies. In some embodiments, antibody sequence elements are fullyhuman, or are humanized, primatized, chimeric, etc, as is known in theart. Moreover, the term “antibody” as used herein, can refer inappropriate embodiments (unless otherwise stated or clear from context)to any of the art-known or developed constructs or formats for utilizingantibody structural and functional features in alternative presentation.For example, in some embodiments, an antibody utilized in accordancewith the present disclosure is in a format selected from, but notlimited to, intact IgG, IgE and IgM, bi- or multi-specific antibodies(e.g., Zybodies®, etc), single chain Fvs, polypeptide-Fc fusions, Fabs,cameloid antibodies, masked antibodies (e.g., Probodies®), Small ModularImmunoPharmaceuticals (“SMIPs™”), single chain or Tandem diabodies (Tand Ab®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTE® s, ankyrinrepeat proteins or DARPINs®, Avimers®, a DART, a TCR-like antibody,Adnectins®, Affilins®, Trans-bodies®, Affibodies®, a TrimerX®,MicroProteins, Fynomers®, Centyrins®, and a KALBITOR®. In someembodiments, an antibody may lack a covalent modification (e.g.,attachment of a glycan) that it would have if produced naturally. Insome embodiments, an antibody may contain a covalent modification (e.g.,attachment of a glycan, a payload (e.g., a detectable moiety, atherapeutic moiety, a catalytic moiety, etc.), or other pendant group(e.g., poly-ethylene glycol, etc.)).

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; triabodies; tetrabodies; linear antibodies;single-chain antibody molecules; and multi-specific antibodies formedfrom antibody fragments. For example, antibody fragments includeisolated fragments, “Fv” fragments (consisting of the variable regionsof the heavy and light chains), recombinant single chain polypeptidemolecules in which light and heavy chain variable regions are connectedby a peptide linker (“ScFv proteins”), and minimal recognition unitsconsisting of the amino acid residues that mimic the hypervariableregion. In many embodiments, an antibody fragment contains sufficientsequence of the parent antibody of which it is a fragment that it bindsto the same antigen as does the parent antibody; in some embodiments, afragment binds to the antigen with a comparable affinity to that of theparent antibody and/or competes with the parent antibody for binding tothe antigen. Examples of antigen binding fragments of an antibodyinclude, but are not limited to, Fab fragment, Fab′ fragment, F(ab′)2fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd′fragment, Fd fragment, and an isolated complementarity determiningregion (CDR) region. An antigen binding fragment of an antibody may beproduced by any means. For example, an antigen binding fragment of anantibody may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively or additionally,antigen binding fragment of an antibody may be wholly or partiallysynthetically produced. An antigen binding fragment of an antibody mayoptionally comprise a single chain antibody fragment. Alternatively oradditionally, an antigen binding fragment of an antibody may comprisemultiple chains which are linked together, for example, by disulfidelinkages. An antigen binding fragment of an antibody may optionallycomprise a multimolecular complex. A functional antibody fragmenttypically comprises at least about 50 amino acids and more typicallycomprises at least about 200 amino acids.

By “antigen” is defined as a molecule that provokes an immune response.This immune response may involve either antibody production, or theactivation of specific immunologically-competent cells, or both. Theskilled artisan will understand that any macromolecule, includingvirtually all proteins or peptides, can serve as an antigen.Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. Moreover, a skilled artisan will understand that anantigen need not be encoded by a “gene” at all. An antigen can begenerated synthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

The term, “autologous” refers to any material derived from the sameindividual to which it is later re-introduced into the individual.

The term “cell therapy antigen” as used herein is meant to refer to oneor more antigens (that are genetically engineered or naturallyoccurring) that can be recognized by effector cells (e.g., geneticallyengineered CAR T cell or genetically engineered or naturally occurringTCR). An antigen that is expressed on tumors (i.e., “tumor-associatedantigen” or TAA) is one type of cell therapy antigen. An antigen that isexpressed selectively on tumors (i.e., “tumor-selective antigen” or TSA)is also a type of cell therapy antigen. An antigen can also be expressedon tumor cells through therapeutic intervention, for example, bytransducing tumor cells, in vivo, with genetic materials of the presentinvention, as described herein.

As used herein, the term “fusion protein” generally refers to apolypeptide including at least two segments, each of which shows a highdegree of amino acid identity to a peptide moiety that (1) occurs innature, and/or (2) represents a functional domain of a polypeptide.Typically, a polypeptide containing at least two such segments isconsidered to be a fusion protein if the two segments are moieties that(1) are not included in nature in the same peptide, and/or (2) have notpreviously been linked to one another in a single polypeptide, and/or(3) have been linked to one another through action of the hand of man.

The term “promoter” refers to a region of a DNA sequence that directsexpression of genes. The promoter is typically upstream from the startof transcription start site and is involved in recognition and bindingof RNA polymerase and other transcription machinery (e.g., otherproteins) to initiate transcription of a polynucleotide sequence.

The term “solid tumor” is meant as an abnormal mass that usually doesnot contain cysts or liquid areas. Solid tumors can be benign ormalignant. Different types of solid tumors are named for the types ofcells that form them (such as sarcomas, carcinomas, and lymphomas).Examples of solid tumors such as sarcomas and carcinoma, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma,and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNStumors (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases.

An “individual” or “subject” can be a vertebrate, a mammal, or a human.Mammals include, but are not limited to, farm animals, sport animals,pets, primates, mice and rats. In one aspect, a subject is a human. An“individual” or “subject” can be a “patient” (e.g., under the care of aphysician) but in some cases, an individual or subject is not a patient.

“Pseudoviruses” or “papilloma pseudoviruses” or “papillomavirus genetransfer vectors” refer to one or more papillomavirus capsid proteinsthat assemble and package heterologous nucleic acids (e.g., DNA) with orwithout viral nucleic acids (e.g., DNA) into infectious particles. Themethods used to produce papilloma pseudoviruses are known in the art andare described, for example, in U.S. Pat. Nos. 6,599,739, 7,205,126, and6,416,945; and in Buck and Thomspon, Production of Papillomavirus-BasedGene Transfer Vectors. Current Protocols in Cell Biology 26.1.1-26.1.19,December 2007, all of which are incorporated by reference.

The term “T cell receptor” or “TCR” refers to a heterodimeric receptorfound on the surface of T lymphocytes. TCRs are antigen-specificmolecules that are responsible for recognizing antigenic peptides of themajor histocompatibility complex (MEW) on the surface of antigenpresenting cells (APCs), or any other nucleated cell. They are membersof the immunoglobulin superfamily, and typically consist of two chains;alpha (α) and beta (β), while a small subset of TCRs are formed byvariable gamma (γ) and delta (δ) chains. The chains pair on the surfaceof a T cell to form a heterodimeric receptor.

The term “transfected” or “transformed” or “transduced” is defined as aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny. In some embodiments, the host cell is a cancer cell, forexample a tumor cell such as solid tumor cell.

The term “tropism” refers to the movement or targeting of a viral vectortowards a receptor.

A “vector” is a composition which comprises an isolated nucleic acid,and which can be used to deliver the isolated nucleic acid to theinterior of a cell. Numerous vectors are known in the art including, butnot limited to, linear polynucleotides, polynucleotides associated withionic or amphiphilic compounds, plasmids, and viruses. Thus, the term“vector” includes an autonomously replicating plasmid or a virus. Theterm should also be construed to include non-plasmid and non-viralcompounds which facilitate transfer of nucleic acid into cells, such as,for example, polylysine compounds, liposomes, and the like. Examples ofviral vectors include, but are not limited to, adenoviral vectors,adeno-associated virus and phage vectors (AAVP), retroviral vectors,human papilloma virus (HPV) pseudovirus vectors, and the like.

II. Compositions

The invention provides for compositions that can be used forintroduction of fusion proteins into cancer cells (e.g., tumor cells)that will make the cancer cells more susceptible for destruction byeither an individual's immune system and/or additional therapeuticagents. Compositions can include, but are not limited to, vectors andvarious constructs described herein, host cells (including tumor cells)containing such vectors and/or constructs, host cells expressing orcapable of expressing these vectors and/or constructs, kits containingthe vectors, constructs, instructions, and/or reagents and the like.

Cancer cells (e.g., tumors) can be transduced with fusion proteins. Asthe fusion proteins are expressed (and/or secreted), the proteins canalso permeate the tumor microenvironment. Non-limiting examples offusion proteins contemplated by the invention include: antibody fusionsso that the antibody or antibody fragment that bind to one or moretumor-associated antigens (TAA) or tumor-specific antigens (TSA),cytokine target fusions, bi-specific T-cell engagers (BITES) or CD19variants.

Vector Design

The nucleic acid sequences coding the desired gene of interest can becloned into a number of types of vectors. For example, the nucleic acidcan be cloned into a plasmid, a phagemid, a phage derivative, an animalvirus, and a cosmid. Other vectors can include expression vectors,replication vectors, probe generation vectors, sequencing vectors, andviral vectors. In other examples, the vector can be a foamy viral (FV)vector, a type of retroviral vector made from spumavirus. Viral vectordesign and technology is well known in the art as described in Sambrooket al, (Molecular Cloning: A Laboratory Manual, 2001), and in othervirology and molecular biology manuals.

Transduction Methods

Transfer of nucleic acid to a cell for gene-modification of the cell inorder for the cell to express a gene of interest is widely performed viatransduction (e.g., viral transduction). The nucleic acid sequencecoding for the desired gene of interest or portion thereof (e.g., tumorassociated antigen) can be obtained using recombinant methods known inthe art. Exemplary methods include screening libraries from cellsexpressing the gene, deriving the gene from a vector, or isolatingdirectly from cells and tissues. These methods are performed usingstandard techniques. In other embodiments, the gene of interest can beproduced synthetically rather than cloned. Gene delivery methods arewell-known in the art, for example, U.S. Pat. No. 5,399,346.

Viral Transduction

Viruses are highly efficient at nucleic acid delivery to specific celltypes, while often avoiding detection by the infected host immunesystem. These features make certain viruses attractive candidates asvehicles for introduction of cellular therapy targets into cancer cells,e.g., solid tumor cells. A number of viral based systems have beendeveloped for gene transfer into mammalian cells. Examples of viralvectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, lentiviruses, poxviruses,herpes simplex 1 virus, herpes virus, oncoviruses (e.g., murine leukemiaviruses), and the like. In general, a suitable vector contains an originof replication functional in at least one organism, a promoter sequence,convenient restriction endonuclease sites, and one or more selectablemarkers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

Lentiviral and Retroviral transduction can be enhanced by the additionof polybrene (SantaCruz sc-134220; Millipore TR-1003-G; Sigma 107689), acationic polymer (also known as hexamehtrine bromide) that is used toincrease the efficiency of the retrovirus transduction.

For example, retroviruses provide a platform for gene delivery systems.Retroviruses are enveloped viruses that belong to the viral familyRetroviridae. Once in a host's cell, the virus replicates by using aviral reverse transcriptase enzyme to transcribe its RNA into DNA. Theretroviral DNA replicates as part of the host genome, and is referred toas a provirus. A selected gene can be inserted into a vector andpackaged in retroviral particles using techniques known in the art. Therecombinant virus can then be isolated and delivered to cells of thesubject either in vivo or ex vivo. A number of retroviral systems areknown in the art, for example See U.S. Pat. Nos. 5,994,136, 6,165,782,and 6,428,953.

Retroviruses include the genus of Alpharetrovirus (e.g., avian leukosisvirus), the genus of Betaretrovirus; (e.g., mouse mammary tumor virus)the genus of Deltaretrovirus (e.g., bovine leukemia virus and humanT-lymphotropic virus), the genus of Epsilonretrovirus (e.g., Walleyedermal sarcoma virus), and the genus of Lentivirus.

In other embodiments, the retrovirus is a lentivirus a genus of virusesof the Retroviridae family, characterized by a long incubation period.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. Lentiviral vectors have anadvantage to other viral vectors in that they can transducenon-proliferating cells and show low immunogenicity. In some examples,the lentivirus includes, but is not limited to human immunodeficiencyviruses (HIV-1 and HIV-2), simian immunodeficiency virus (S1V), felineimmunodeficiency virus (FIV), equine infections anemia (EIA), and visnavirus. Vectors derived from lentiviruses offer the means to achievesignificant levels of gene transfer in vivo.

In embodiments, the vector is an adenovirus vector. Adenoviruses are alarge family of viruses containing double stranded DNA. They replicatethe DNA of the host cell, while using the host's cell machinery tosynthesize viral RNA DNA and proteins. Adenoviruses are known in the artto affect both replicating and non-replicating cells, to accommodatelarge transgenes, and to code for proteins without integrating into thehost cell genome.

In some embodiments, an AAVP vector is used. The AAVP vector is a hybridof prokaryotic-eukaryotic vectors, which are chimeras of geneticcis-elements of recombinant adeno-associated virus and phage. An AAVPcombines selected elements of both phage and AAV vector systems,providing a vector that is simple to produce in bacteria with nopackaging limit, while allowing infection of mammalian cells combinedwith integration into the host chromosome. Vectors containing many ofthe appropriate elements are commercially available, and can be furthermodified by standard methodologies to include the necessary sequences.Among other things, AAVPs do not require helper viruses or trans-actingfactors. In addition, the native tropism of AAV for mammalian cells iseliminated since there is not AAV capsid formation. Other methods anddetails are in U.S. Pat. No. 8,470,528 and Hajitou A. et al., Cell, 125:358-398, both of which are incorporated herein by reference.

In other aspects, a human papilloma (HPV) pseudovirus is used. Recentstudies have shown that DNA plasmids can be packaged into papillomavirusL1 and L2 capsid protein to generate pseudovirion that can efficientlydeliver DNA. The encapsulation protects the DNA from nucleases andprovides a targeted delivery with great stability. Many of the safetyconcerns associated with the use of viral vectors are mitigated with theHPV pseudoviros because its construct is different from the natural HPVviral genome. Other methods and examples are in Hung, C., et al., PlosOne, 7:7(e40983); 2012, U.S. Pat. No. 8,394,411, and Kines, R., et alInt J of Cancer, 2015, all of which are incorporated herein byreference.

In some aspects, an oncolytic virus is used. Oncolytic virus therapyselectively replicates the virus in cancer cells, and subsequentlyspreads within a tumor without affecting normal tissue. Alternatively,the oncolytic virus preferentially infects and kills cells withoutcausing damage to normal tissues. Oncolytic viruses are also effectiveat inducing immune responses to themselves as well as to the infectedtumor cell. Typically, oncolytic viruses fall into two classes: (I)viruses that naturally replicate preferentially in cancer cells and arenonpathogenic in humans. Exemplary class (I) oncolytic viruses includeautonomous parvoviruses, myxoma virus (poxvirus), Newcastle diseasevirus (NDV; paramyxovirus), reovirus, and Seneca valley virus(picornavirus). A second class (II), include viruses that aregenetically manipulated for use as vaccine vectors, including measlesvirus (paramyxovirus), poliovirus (picornavirus), and vaccinia virus(poxvirus). Additionally, oncolytic viruses may include thosegenetically engineered with mutations/deletions in genes required forreplication in normal but not in cancer cells including adenovirus,herpes simplex virus, and vesicular stomatitis virus. Oncolytic virusescan be used as a viral transduction method due to their low probabilityof genetic resistance because they can target multiple pathways andreplicate in a tumor-selective method. The viral dose within the tumorcan increase with time due to in situ viral amplification (as comparedto small molecule therapies which decrease with time), and safetyfeatures can be built in (i.e., drug and immune sensitivity).

Integration

In some aspects of the invention, the nucleic acids encoding cellulartherapy target(s) or tumor-associated antigen(s) are integrated as partof the tumor cells' genetic makeup. Without being bound by theory,integration of the nucleic acid encoding cellular therapy target(s) canbe helpful in that as the tumor cell replicates, progeny tumor cellswould express the cellular therapy target(s) and, as such, besusceptible to the effector cells and other therapeutic agents(including combination therapy agents). It follows that in someindications, such as metastatic disease, the state of rapid tumor cellproliferation becomes useful in propagating the therapeutics of thepresent invention.

In one embodiment, integration can be achieved by using viruses thatnaturally integrate into the host cell. Integration is a crucial step inreplication of retroviruses as it is a virus genome that has beenintegrated into the DNA of a host cell. Integration is not part of theviral replication cycle, but it can occasionally occur. The virus doesnot directly make new DNA copies of itself while integrated into thehost genome; alternatively, it is passively replicated along with thehost genome and passed on to the original cell's offspring. Integrationof the viral DNA results in permanent insertion of the viral genome intothe host chromosomal DNA, referred as a provirus in the case ofretroviruses.

In other embodiments, integration can be achieved by commerciallyavailable kits, including the CompoZr® Targeted Integration Kit which isdesigned to integrate a gene of interest into the adeno-associated virusintegration site 1 (AAVS1) on human chromosome 19.

In addition to the expression of TAA, the cancer cells (e.g., solidtumor cells) can be engineered so that they also express one or morefusion protein of an antibody or antibody fragment (e.g., scFv) coupledto an antigen that an effector cell will recognize. The antibody orantibody fragment recognizes the tumor cell and binds and presents theantigen for an effector cell to recognize. In one non-limiting example,a solid tumor cell can be transduced with a fusion protein that includesan anti-tumor TAA scFv that binds to a tumor cell, and also part or allof the human CD19 protein. The scFv portion binds to the tumor cell andthe CD19 is presented to effector cells for them to target the tumorcell and destroy the tumor cell. The effector cells can be naturallyoccurring or genetically engineered.

Expressed Gene Approach for Solid Tumor Targeting

Genes productively introduced into tumor cells will provide improvedcellular therapeutic activity by addressing or circumventing criticalbarriers to efficacy. These genes will improve therapeutic efficacy by“propagating” the anti-tumor response, optimize cytokine support in thelocal environment, reverse local immunosuppression, improve cellulareffector functions, and promote cellular access to tumors. Any gene canbe included in an expressed construct as described herein, and thepresent disclosure is not limited to any particular gene. Exemplary,non-limiting types of genes that can be included as cellular therapytargets include, e.g., targets for additional cellular therapeutics,polypeptide antigens, antibodies, cytokines, agents targeting tumormicroenvironment, and agents supporting immune cellgrowth/proliferation.

An expressed gene contains a promotor with its associated elements, anda gene sequence. An expressed gene contains three essentialcharacteristics: 1) an optimal promoter for expression in the tumorcell, 2) an optimal expressed gene sequence, and 3) an optimizedexpression pattern with defined kinetics.

A set of promoters is developed to drive diverse expression patterns.Examples include; rapid and sustained expression, measured in days, orrapid but reversible expression, or delayed expression. Also, the levelof expression can be modified by selective use of regulatory andpromoter elements. Such methods are well understood, for example, E. D.Papadakis, et al., Current Gene Therapy, 4: 89-113 (incorporated hereinby reference).

III. Methods for Treating Cancer

The invention provides for compositions and methods for treating cancerby engineering a system whereby the cancer cells (e.g., tumor cells orsolid tumor cells) secrete fusion proteins that include TAA or TSA and atherapeutic agent. An individual having cancer or suspected of havingcancer can be given a composition that allows for the in vivotransduction of their cancer cells. The administration can be by anymeans, including, but not limited to intravenously, systemically,intramuscularly, intraperitonally, or intra-tumoral injection.

Targets for Additional Cellular Therapeutics

In some embodiments, the cell therapy antigen that is expressed on localtumor cells following transduction and secretion of fusion proteins isrecognized by the effector cells, and can comprise a tumor associatedantigen (TAA). In one embodiment, TAA expression can be restricted tothe tumor cell population alone, expressed by all tumor cells, andexpressed on the tumor cell surface. Other antigens are overexpressed ontumor cells, but may be found on normal cells at lower levels ofexpression and thus are tumor-selective antigens (TSA). In addition,some tumor antigens arise as “passenger mutations”, i.e. arenon-essential antigens expressed by tumor cells that have defectivecontrol over DNA repair, thus accumulating mutations in diverseproteins. Some tumor antigens are proteins that are produced by tumorcells that elicit an immune response; particularly T-cell mediatedimmune responses. Tumor-specific molecules that may be targeted bycellular therapy targets may include tumor associated antigens wellknown in the art and can include, for example, a glioma-associatedantigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1,MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS),intestinal carboxyi esterase, mut hsp70-2, M-CSF, prostase,prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumorantigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22,insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.The application of technologies, including next generation sequencing(NGS) of tumor genomes and exomes, and high-sensitivity massspectrometry (protein sequencing) analysis of the tumor proteome, iscontinuing to identify novel TAA and TSA antigens of use for thisinvention.

NGS is a method of high-throughput sequencing that performs massivelyparallel sequencing, during which millions of fragments of DNA from asingle sample are sequenced in unison. NGS facilitates high-throughputsequencing, which allows an entire genome to be sequenced in less thanone day. The creation of NGS platforms has made sequencing accessible tomore labs, rapidly increasing the amount of research and clinicaldiagnostics being performed with nucleic acid sequencing, and thus haverevolutionized genomics and molecular biology. Some NGS technologiesinclude Illumina (Solexa) sequencing, Roche 454 sequencing, Ion torrent:proton/PGM sequencing, and SOLiD sequencing.

An alternative method for identifying tumor specific antigens is directprotein sequencing. Protein sequencing of enzymatic digests usingmultidimensional MS techniques (MSn) including tandem mass spectrometry(MS/MS)) can also be used to identify antigens. Such proteomicapproaches permit rapid, highly automated analysis (See, e.g., K.Gevaert and J. Vandekerckhove, Electrophoresis 21: 1145-1154 (2000,incorporated herein by reference)). Furthermore, high-throughput methodsfor de novo sequencing of unknown proteins may be used to analyze theproteome of a patient's tumor to identify expressed antigens. Forexample, meta shotgun protein sequencing may be used to identifyexpressed antigens (See e.g., Guthals et al. (2012), Molecular andCellular Proteomics 11(10): 1084-96, incorporated herein by reference).

Non-limiting examples of tumor antigens that can used include EphA2,HER2, GD2, Glypican-3, 5T4, 8H9, αvβ6 integrin, BCMA, B7-H3, B7-H6,CAIX, CA9, CD19, CD20, CD22, kappa light chain, CD30, CD33, CD38, CD44,CD44v6, CD44v7/8, CD70, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFRvIII,EGP2, EGP40, EPCAM, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, fetal AchR,Folate Receptor a, GD2, GD3, HLA-AI MAGE A1, HLA-A2, IL11Ra, IL13Ra2,KDR, Lambda, Lewis-Y, MCSP, Mesothelin, Mud, Muc16, NCAM, NKG2D ligands,NY-ESO-1, PRAME, PSCA, PSC1, PSMA, ROR1, SURVIVIN, TAG72, TEM1, TEM8,VEGRR2, carcinoembryonic antigen, HMW-MAA, and VEGF receptors. Otherexemplary antigens that can be used are antigens that are present within the extracellular matrix of tumors, such as oncofetal variants offibronectin, tenascin, or necrotic regions of tumors.

Additional tumor-selective molecules can be used include any membraneprotein or biomarker that is expressed or overexpressed in tumor cellsincluding, but not limited to, integrins (e.g., integrin αvβ3, α5β1),EGF Receptor Family (e.g., EGFR2, Erbb2/HER2/neu, Erbb3, Erbb4),proteoglycans (e.g., heparan sulfate proteoglycans), disialogangliosides(e.g., GD2, GD3), B7-H3 (aka CD276), cancer antigen 125 (CA-125),epithelial cell adhesion molecule (EpCAM), vascular endothelial growthfactor receptors 1 and 2 (VEGFR-1, VEGFR-2), CD52, carcinoembryonicantigen (CEA), tumor associated glycoproteins (e.g., TAG-72), cluster ofdifferentiation 19 (CD19), CD20, CD22, CD30, CD33, CD40, CD44, CD74,CD152, mucin 1 (MUC1), tumor necrosis factor receptors (e.g., TRAIL-R2),insulin-like growth factor receptors, folate receptor a, transmembraneglycoprotein NMB (GPNMB), C-C chemokine receptors (e.g., CCR4), prostatespecific membrane antigen (PSMA), recepteur d'origine nantais (RON)receptor, cytotoxic T-lymphocyte antigen 4 (CTLA4), and other tumorspecific receptors or antigens.

As exemplified by the non-limiting examples of TAA and TSA targetsrecited herein, there is a large and diverse selection of antigens towhich one of skill in the art can direct the transduced and expressedtherapeutics described herein. For example, the therapeutics mightcomprise a secreted fusion protein that contains a scFv antigen bindingdomain (e.g. anti-MUC16, anti-CEA, anti-PSMA) fused to the targetantigen (e.g. CD19) that can be recognized by a CAR T cell. In thisembodiment, the secreted fusion protein has two functional domains, thescFv that binds to the target tumor cell surface, and the CD19 proteindomain that is presented as a target for the CAR T cell (FIG. 1). Itwill be immediately apparent that the scFv can be selected to target oneof many diverse antigens, and that the protein domain to be recognizedby the cellular therapeutic (also, the “effector cell” of the presentinvention), can also be diverse, e.g. recognized by a specific CAR Tcell, or a TCR T cell, or a characterized TIL or an NK cell. It will befurther recognized that modern antibody engineering allows the use ofbispecific recognition to be built into the scFv portion of thetherapeutic, such that effective binding to the tumor cells isaccomplished only when both arms of a bispecific scFV can bind. Therange of bispecific technologies available is broad and the molecularbiology and protein chemistry tools and principles required foreffective bispecific antibody engineering are well understood, see forexample, Kontermann, R. MAbs 2012; 4(2) 182-97 (incorporated herein byreference).

Further, multiple genes can be encoded in a single CAR expressionconstruct by using, for example, in frame or independent IRES initiationsites for individual elements. Alternatively, inducible methods havebeen described, whereby the application of a small molecule can induceor block expression of one or more CAR elements. A wide variety of suchmethods are disclosed, such as inhibitory CARs, costimulatory CARs,“cideCARs”, on switches and others, the use of which can further modifyor alter the activity of CAR T cells (see Baas, T. SciBX 7(25);doi:10.1038/scibx.2014.725).

Antibody-Drug Conjugate Targets

In other embodiments, the transduced tumor cells secrete fusion proteinsthat are targeted by antibody-drug conjugates that are known andinclude, e.g., brentuximab vedotin (ADCETRIS, Seattle Genetics);trastuzumab emtansine (Roche); Gemtuzumab ozogamicin (Pfizer); CMC-544;SAR3419; CDX-011; PSMA-ADC; BT-062; CD30, HER2, and IMGN901 (see, e.g.,Sassoon et al., Methods Mol. Biol. 1045:1-27 (2013); Bouchard et al.,Bioorganic Med. Chem. Lett. 24: 5357-5363 (2014)). In other embodiments,the transduced tumor cells secrete fusion protins which can be targetedby antibodies having antibody dependent cellular cytotoxicity functionsuch as those recognized by rituximab, ocrelizumab, ipilimumab,cituximab, erbitux and many others. Accordingly, in some embodiments, anucleic acid encoding a polypeptide antigen as part of the fusionprotein that binds to one or more of such known antibody-drug conjugatescan be included in as a cellular therapy target described herein.

Cytokine Fusion Proteins

In some embodiments, the tumor and tumor microenvironment is transducedwith a cytokine fusion protein, e.g., a fusion protein of a cytokine(e.g., an anti-tumor cytokine) and a target for one or more additionalcellular therapeutics described herein (e.g., a CAR-T target). Exemplarycytokines may bind to the tumors, and present the tumor with thecytokine. For example, some contemplated targets include CD19, CD20,CD21, CD22, CD24, and BCMA. Such a cellular therapy can provide both atarget for one or more additional cellular therapeutics (e.g., a CAR-Ttarget) and a stimulatory cytokine at a tumor surface. For example, anexpressed and/or secreted construct can encode a cytokine-CD19 fusionprotein, or a fusion of a cytokine and a CD19 fragment, e.g., a CD19fragment to which a CD19-CAR-T cell binds. In some embodiments, a CD19fragment is a CD19 IgC domain. Without wishing to be bound by theory, asingle expressed construct encoding such a fusion protein advantageouslyallows a cellular therapeutic to be genetically engineered using aminimal (e.g., a single) transgene. An additional benefit of usingcytokine fusion proteins is to utilize their tight binding to theirreceptors, in addition to the cytokine functional effect.

In some embodiments, one or more cytokines secreted as part of acytokine fusion protein bind to cells at high affinity (e.g., KD ofabout 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or less) and/or have lowinternalization rates (e.g., less than about 10, 10², 10³, 10⁴, or 10⁵cytokine molecules per cell per day). Binding affinity andinternalization rates of various cytokines are known in the art and/orcan be measured using known methods.

Pro-Immune Response Agents

In some embodiments, the recombinant tumor described herein encodes, asa fusion protein as described herein, one or more pro-immune responseagents, e.g., one or more cytokines used in cancer therapy.Non-limiting, exemplary cytokines that can be included include, e.g.,IFNα, IFNβ, IFNγ, IL-1, IL-2, IL-7, IL-12, IL-15, IL-21, IL-36, TNF,LTα, GM-CSF, G-CSF, a TLR agonist, and an immune checkpoint antibodyfragment.

Known problems associated with cytokine therapy include, e.g., high doserequirements, toxicity, and limited efficacy. Thus, in some embodiments,expressed constructs are used to deliver one or more cytokines at aspecific site and/or at a specific dose (e.g., to reduce or eliminateone or more risks associated with cytokine therapy).

In some embodiments, expression of a cytokine (e.g., animmunostimulatory cytokine) at or near a surface of a tumor induces animmune response to the tumor. In some embodiments, an expressed cytokinefusion protein can be a target for one or more additional cellulartherapeutics (e.g., one or more additional CAR-T cells). In someembodiments, secretion of a cytokine fusion protein near a surface of atumor induces an immune response to the tumor and is also used as atarget for one or more additional cellular therapeutics (e.g., one ormore additional CAR-T cells). An example is an Interferon alpha cytokinefused to the CD19 protein domain recognized by a CAR T cell with CD19reactivity. The IFNalpha molecule binds with high affinity to interferonreceptors on or near the tumor cell, thus supporting cellulartherapeutic activity directed to CD19. In another example, theinterferon alpha cytokine is fused to a scFV that recognizes a TAA or aTSA on the same tumor cell type, thus binding back on to the cell andsurrounding cells, and inducing or supporting an IFNalpha-driven immuneresponse.

For example, release of IL-21 can be used to induce expansion and/oreffector differentiation of CD8+ T cells and/or support NK cellactivation and cytolytic activity. In one exemplary method, an expressedconstruct encodes IL-21. Upon binding of a scFv directed an antigen on atumor cell, a cellular therapeutic described herein exhibits prolongedrelease of IL-21. Exemplary cellular therapeutics include, e.g., CAR-Tcells, CAR-NK cells, TCR-T cells, TIL cells, allogenic NK cells, andautologous NK cells.

In another exemplary method, induced release of IL-15 fusion proteinscan be used to support NK cell expansion and/or to recruit NK cells topromulgate an anti-tumor response and to support the survival andexpansion of cellular therapeutics. Exemplary cellular therapeuticsinclude, e.g., CAR-T cells, CAR-NK cells, TCR-T cells, TIL cells,allogenic NK cells, and autologous NK cells. In this example a scFv thatrecognizes a TAA or a TSA on the target tumor cell type binds, andpresents IL-15 in the local environment. In a further exemplification,of relevance to all of the cytokine examples proposed, the secretedfusion protein is trifunctional, having a scFV that recognizes a TAA ora TSA on the target tumor cell type, a cytokine encoded into a beta loopor beta strand within the heavy or light chain variable region that isnot engaged in antigen binding, and expressing a target antigen for thebinding of a cellular therapeutic. The utilization of CDRs within theheavy and light variable domains of a scFv is readily determined fromthe sequence of the CDR as well as through the use of databases thatindicate which residues are involved in antigen engagement and which arenot involved but are otherwise solvent exposed, i.e. useful forexpression of an encoded sequence such as a cytokine.

Cell Recruiting Moieties

In some examples, the recombinant tumor cells can express, as part of afusion protein, scFv or TCR that may be fused to cell recruitingmoieties (e.g., anti-CD3 or anti-CD16). In other embodiments, bispecificT cell engager (BiTE®) technology is utilized to help engage the body'sendogenous T cells and to target cancer cells that have been engineeredto express one or more TAA's. The antibody constructs of the BiTE®technology are constructed by genetically linking the minimal bindingdomains of monoclonal antibodies for TAA or tumor-associated surfaceantigens and for the T cell receptor-associated molecule, onto a singlepolypeptide chain. One antibody is specific for a selected surfaceantigen on a targeted tumor cell, and the other antibody is specific formoiety (e.g., CD3), tied to the T-cell receptor complex on the surfaceof T cells. The BiTE® technology binds polyclonal cytotoxic T cells andtargeted malignant cells.

Polypeptide Antigens

In some embodiments, a target for one or more additional cellulartherapeutics is or comprises a polypeptide antigen. The polypeptideantigen to be expressed by an expressed construct, and is not limited toany particular polypeptide or portion thereof, provided that anadditional cellular therapeutic (e.g., CAR-T cell) can be engineered torecognize and bind to such polypeptide target. In some embodiments, thepolypeptide target is a polypeptide that is not a tumor-associatedantigen. In some embodiments, the target is a tumor antigen, e.g., BCMA,CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met, PSMA, GlycolipidF77, EGFRvIII, GD-2, NY-ESO-1 TCR, or MAGE A3 TCR.

Antibody Fusion Proteins

In some embodiments, the tumor and tumor microenvironment is transducedwith a fusion protein comprising an antibody (or antigen-bindingfragment thereof, e.g., secreted scFv or other antibody formats) and atarget for one or more additional cellular therapeutics (e.g., a CAR-Ttarget). An antibody (or fragment) can be selected to bind, e.g., to atumor antigen (e.g., a tumor antigen described herein), and its fusionpartner can include a target for one or more additional cellulartherapeutics. Such antibodies (or antigen-binding fragments) include,e.g., a monoclonal antibody (mAb), including, for example, scFv and fulllength mAbs, a VHH domain, a diabody, a nanobody, etc. In one example, aconstruct encodes a secreted fusion protein consisting of a mAb (e.g.,an anti-tumor associated antigen mAb or antigen-binding fragment) andCD19 or a fragment thereof (e.g., a CD19 Ig domain).

In some embodiments, an antibody (or fragment) binds to an antigenexpressed on several types of cells. In some embodiments, an antibody(or fragment) binds to a tumor-selective antigen. In some embodiments,an antibody (or fragment) binds to a tumor-selective, but not specific,antigen. In some embodiments, an antibody (or fragment) binds to a tumorantigen associated with a hematologic malignancy. In some embodiments,an antibody (or fragment) binds to a tumor antigen associated with asolid tumor. In some embodiments, an antibody (or fragment) binds to oneor more of BCMA, CD19, CD20, CD22, ROR1, mesothelin, CD33/IL3Ra, c-Met,PSMA, Glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, and MAGE A3 TCR.

The target, which is placed on the tumor cells, can be CD19. Other Bcell targets can be used, including but not limited to CD20, CD21, CD22,CD23, CD24, CD72, CD79a, CD79b, and BCMA. These B cell targets haveparticular advantages as CAR T cell targets, along with the list ofother targets. In addition, the target can include CD30, Her 2, a targetfor ADC's or radioimmunotherapy (e.g., a monoclonal antibody carrying aradioisotope).

Peptides that Inhibit Local (e.g., Tumor Microenvironment) Factors

In some embodiments, peptides (e.g., polypeptides and fragments thereof)that inhibit local factors can be expressed as a fusion protein by thetumor cell. Nucleic acids coding for these peptides can be engineered asdescribed herein and/or by any method known to one of skill in the artsuch that the peptide(s) are expressed. Non-limiting examples includeTGFbeta, adenosine receptor 2, vascular endothelial growth factor(VEGF), platelet-derived growth factor (PDGF), indoleamine2,3-dioxygenase 1 (IDO1), and matrix metalloproteinases (MMPs)).

CD19 as a Scaffold for Inducible CD19 Variant Proteins and CD19 VariantFusion Proteins

CD19 is a 95 kd transmembrane glycoprotein belonging to the Igsuperfamily and includes two extracellular C2-type Ig domains (see,e.g., Tedder Nature Rev. Rheum. 5:572-577 (2009); Wang et al., Exp.Hematol. Oncol. 2012 Nov. 29; 1(1):36. doi: 10.1186/2162-3619-1-36.)).In some embodiments, one or both of the C2-type Ig domains are used asscaffolds for mutagenesis, and CD19 variants (e.g., CD19 or a portionthereof that include one or more mutations within one or both C2-type Igdomains) can be screened and selected for binding to a target antigendescribed herein.

The nucleotide sequence of human CD19 is known (see Genbank AccessionNo. M84371.1). To provide variant nucleic acid sequences that encodeCD19 variants that bind a particular antigen, a number of methods knownin the art may be utilized. In some embodiments, a screening procedureis used that enables identification and/or isolation of nucleic acidsthat encode CD19 variants that bind a particular antigen. Exemplarymethods include a so-called biopanning step, known from technologiessuch as phage display (Kang, A. S. et al. 1991. Proc Natl Acad Sci USA88, 4363-4366), ribosome display (Schaffitzel, C. et al. 1999. J.Immunol. Methods 231, 119-135), DNA display (Cull, M. G. et al. 1992.Proc Natl Acad Sci USA 89, 1865-1869), RNA-peptide display (Roberts, R.W., Szostak, J. W., 1997. Proc Natl Acad Sci USA 94, 12297-12302),covalent display (WO 98/37186), bacterial surface display (Fuchs, P. etal. 1991. Biotechnology 9, 1369-1372), yeast surface display (Boder, E.T., Wittrup, K. D., 1997. Nat Biotechnol 15, 553-557) and eukaryoticvirus display (Grabherr, R., Ernst, W., 2001. Comb. Chem. HighThroughput. Screen. 4, 185-192). FACS and magnetic bead sorting are alsoapplicable for enrichment (panning) purposes using labeled antigen.Immunodetection assays such as ELISA (Dreher, M. L. et al. 1991. J.Immunol. Methods 139, 197-205) and ELISPOT (Czerkinsky, C. C. et. al.1983. J Immunol Methods. 65, 109-21) can also be used either following abiopanning step or alone.

Thus, in some embodiments, an inducible construct described hereinencodes a CD19 variant (or fragment), either alone or as part of afusion protein described herein. For example, an inducible constructdescribed herein can encode a CD19 variant (or fragment) selected tobind to a tumor agent and which, upon expression, can bind to the tumorantigen and that itself can be a target for an additional cellulartherapeutic (e.g., a CAR-T cell that binds CD19). In some embodiments,an inducible construct described herein encodes a CD19 variant thatincludes a C2-type Ig domain variant selected to bind a tumor antigen.Upon expression of the CD19 variant, the C2-type Ig domain binds to thetumor antigen on a tumor cell. Subsequently, treatment with (e.g.,administration to a subject of) a CAR-T cell that recognizes CD19 killsthe tumor cell to which the CD19 variant is bound. In some embodiments,an inducible construct described herein encodes a CD19 variant thatincludes variants of both C2-type Ig domains, each of which is selectedto bind a tumor antigen (e.g., different epitopes of the tumor antigen).Upon expression of the CD19 variant, the C2-type Ig domains bind to thetumor antigen on a tumor cell. Subsequently, treatment with (e.g.,administration to a subject of) a CAR-T cell that recognizes CD19 killsthe tumor cell to which the CD19 variant is bound.

In some embodiments, a CD19 variant selected for binding to a targetantigen is included in a fusion protein. For example, a CD19 variantthat includes a C2-type Ig domain variant selected to bind a tumorantigen can be fused to an antibody or fragment thereof that also bindsto the tumor antigen (e.g., to a different epitope on the tumorantigen). Exemplary fusion proteins include, e.g., CD19 variant/scFvfusion proteins and CD19 variant/VHH fusion proteins. An inducibleconstruct described herein can encode such a CD19 variant/antibodyfusion protein and upon expression, the CD19 variant and the antibody ofthe fusion protein bind to the tumor antigen on a tumor cell.Subsequently, treatment with (e.g., administration to a subject of) aCAR-T cell that recognizes CD19 kills the tumor cell to which the CD19variant/antibody fusion protein is bound. In some embodiments, asdescribed herein, the CD19 scaffold genes that are useful in the contextof inducible expression will also be useful when modified for theproduction in vitro of soluble, purified fusion proteins. Additional,non-limiting examples of fusion proteins that include CD19 variants (orfragment) as a scaffold include, e.g., CD19 variant/cytokine fusionproteins and CD19 variant/TLR agonist fusion proteins.

Additional, non-limiting examples of fusion proteins that include CD19(or fragment) as a scaffold include, e.g., CD19-cytokine fusionproteins, CD19-TLR agonist fusion proteins. Other B cell restricted cellsurface markers which contain immunoglobulin-like domains include CD22,CD79a and CD79b. These Ig domains can also be mutagenized to generatevariants binding TAA's and TSA's.

Polypeptide Antigens and Antibodies

The below Table 1 presents a non-comprehensive list of certain humanpolypeptide antigens targeted by known, available antibody agents, andnotes certain cancer indications for which the antibody agents have beenproposed to be useful:

TABLE 1 Antibody (commercial or Human Antigen scientific name) Cancerindication CD2 Siplizumab Non-Hodgkin's Lymphoma CD3 UCHT1 Peripheral orCutaneous T-cell Lymphoma CD4 HuMax-CD4 CD19 SAR3419, MEDI-551 DiffuseLarge B-cell Lymphoma CD19 and CD3 or Bispecific antibodies such asNon-Hodgkin's Lymphoma CD22 Blinatumomab, DT2219ARL CD20 Rituximab,Veltuzumab, B cell malignancies (Non-Hodgkin's Tositumomab, Ofatumumab,lymphoma, Chronic lymphocytic leukemia) Ibritumomab, Obinutuzumab, CD22(SIGLEC2) Inotuzumab, tetraxetan, CAT- Chemotherapy-resistant hairy cellleukemia, 8015, DCDT2980S, Bectumomab Hodgkin's lymphoma CD30Brentuximab vedotin CD33 Gemtuzumab ozogamicin Acute myeloid leukemia(Mylotarg) CD37 TRU-016 Chronic lymphocytic leukemia CD38 DaratumumabMultiple myeloma, hematological tumors CD40 Lucatumumab Non-Hodgkin'slymphoma CD52 Alemtuzumab (Campath) Chronic lymphocytic leukemia CD56(NCAM1) Lorvotuzumab Small Cell Lung Cancer CD66e (CEA) LabetuzumabBreast, colon and lung tumors CD70 SGN-75 Non-Hodgkin's lymphoma CD74Milatuzumab Non-Hodgkin's lymphoma CD138 (SYND1) BT062 Multiple MyelomaCD152 (CTLA-4) Ipilimumab Metastatic melanoma CD221 (IGF1R) AVE1642,IMC-A12, MK-0646, Glioma, lung, breast, head and neck, R150, CP 751871prostate and thyroid cancer CD254 (RANKL) Denosumab Breast and prostatecarcinoma CD261 (TRAILR1) Mapatumumab Colon, lung and pancreas tumorsand CD262 (TRAILR2) HGS-ETR2, CS-1008 haematological malignancies CD326(Epcam) Edrecolomab, 17-1A, IGN101, Colon and rectal cancer, malignantascites, Catumaxomab, Adecatumumab epithelial tumors (breast, colon,lung) CD309 (VEGFR2) IM-2C6, CDP791 Epithelium-derived solid tumorsCD319 (SLAMF7) HuLuc63 Multiple myeloma CD340 (HER2) Trastuzumab,Pertuzumab, Ado- Breast cancer trastuzumab emtansine CAIX (CA9) cG250Renal cell carcinoma EGFR (c-erbB) Cetuximab, Panitumumab, Solid tumorsincluding glioma, lung, breast, nimotuzumab and 806 colon, and head andneck tumors EPHA3 (HEK) KB004, IIIA4 Lung, kidney and colon tumors,melanoma, glioma and haematological malignancies Episialin EpitumomabEpithelial ovarian tumors FAP Sibrotuzumab and F19 Colon, breast, lung,pancreas, and head and neck tumors HLA-DR beta Apolizumab Chroniclymphocytic leukemia, non- Hodkin's lymphoma FOLR-1 Farletuzumab Ovariantumors 5T4 Anatumomab Non-small cell lung cancer GD3/GD2 3F8, ch14.18,KW-2871 Neuroectodermal and epithelial tumors gpA33 huA33 Colorectalcarcinoma GPNMB Glembatumumab Breast cancer HER3 (ERBB3) MM-121 Breast,colon, lung, ovarian, and prostate tumors Integrin αVβ3 EtaracizumabTumor vasculature Integrin α5β1 Volociximab Tumor vasculature Lewis-Yantigen hu3S193, IgN311 Breast, colon, lung and prostate tumors MET(HGFR) AMG 102, METMAB, Breast, ovary and lung tumors SCH900105Mucin-1/CanAg Pemtumomab, oregovomab, Breast, colon, lung and ovariantumors Cantuzumab PSMA ADC, J591 Prostate Cancer PhosphatidylserineBavituximab Solid tumors TAG-72 Minretumomab Breast, colon and lungtumors Tenascin 81C6 Glioma, breast and prostate tumours VEGFBevacizumab Tumour vasculature

Accordingly, a cellular therapeutic that targets an expressed constructencoding a polypeptide antigen can be used in combination with one ormore of these (or other) known antibodies.

The following examples are provided for illustrative purposes only andare not intended to limit the invention in any manner.

EXEMPLIFICATION Example 1: An Antigen-Activation Controlled PromoterPromotes Cytokine Release after Cellular Therapeutic Cells Encounter anAntigen (e.g., Tumor Cells or Their Local Environment)

Cytokine support for cancer therapeutics has a long history (e.g. thesystemic use of TNF, LTa, IFNa and IL-2). Inherent problems withsystemic cytokine therapy include high dosage requirement, minimalefficacy, and toxicity (lethal in the case of TNF and LTa). Suchtoxicity limits the use of IL-12 and IL-15.

For example, systemic recombinant IL-12 induced multiple serious adverseeffects, including renal and systemic toxicity. High-dose levels werelinked to temporary immune suppression, which would be unfavorable foreffective immunotherapy. (See, Leonard et al., Blood 1997; 90:2541-8,and Colombo, M P et al., CytokineGrowth Factor Rev 2002; 13:155-68,incorporated herein by reference. The majority of systemic IL-12 trialswere associated with toxic adverse events and limited efficacy, sincethe cytokine did not reach the tumor site(s) in sufficient concentration(S Tugues, et al., 2015 Cell Death Differ 22: 237-246, incorporatedherein by reference.

Recombinant IL-15 induces NK and CD8 cell mediated toxicities.Dose-limiting toxicities observed in patients receiving rIL-15 includedgrade 3 hypotension, thrombocytopenia, and elevations of ALT and AST,resulting in a suboptimal maximum-tolerated dose, having minimalclinical efficacy. (See, Conlon, K., et al., J. of Clinical OncologyJan. 1, 2015: 74-82, incorporated herein by reference).

These and similar studies illustrate the dose-limiting toxicity andminimal efficacy associated with the uncontrolled administration andsystemic distribution of cytokines. Accordingly, an individual withcancer or suspected of having cancer (e.g., tumor) is administered witha composition that allows for the direct transduction of the tumor cellsso that a fusion protein of a cytokines and a target. The administrationcan be intravenously or intra-tumorally.

Example 2: Release of Cytokines From Transduced Tumor Cells

CAR T cells, CAR NK cells, TCR T cells, TIL, allogeneic NK cells andautologous NK cells are engineered for prolonged release of IL-21 as acytokine fusion protein with TAA. Expansion and effector differentiationis induced in CD8+ T cells, and NK cell activation and cytolyticactivity is supported.

Engagement of activation signals rapidly secretes IL-15 under thecontrol of the TNF promoter, which supports NK cell expansion (e.g., CARNK, allogeneic NK cells, and autologous NK cells) or recruits NK cellpromulgation in an anti-tumor response (e.g., CAR T, TCR, TIL).Alternatively, various combinations of cytokines expressed and promotersare used, and are expressed in a suitable manner (IL-2, IL-12, IL-36g,IFNg) as a fusion protein.

Sustained local production of an anti-TAA scFv antibody fused to IL-12recruits immune cells to support and expand the anti-tumor immuneresponse triggered by engagement with the CAR T cell or other cellulartherapeutic.

Example 3: The Targeting Method Utilizes a Single Chain VariableFragment (scFv) Antibody (or Fragment Thereof), or Secreted HeterodimerTCR Alpha/Beta or Gamma/Delta Chains are Fused to an Antibody-DrugConjugate Target

The target is the polypeptide sequence, protein domain or domainsrecognized by the ADC, which is an antibody coupled to a toxin. ADCtargets are the HER2 receptor, the CD30 cell surface protein, folatereceptor alpha, and CD19, among many others.

The targeting method utilizes an scFv antibody or fragment thereof, andis fused to an antibody-drug conjugate target. Alternatively, a secretedheterodimer TCR alpha/beta or gamma/delta chain targeting method isused. Example antibody-drug conjugate target includes CD30, HER2, orADCC antibody targets.

Example 4: The Targeting Method Utilizes a Single Chain VariableFragment (scFv) Antibody (or Fragment Thereof), or Secreted HeterodimerTCR Alpha/Beta or Gamma/Delta Chains are Fused to a Pro-Immune ResponseAgent

A “pro-immune response agent” is any agent capable of stimulating theimmune response by stimulation cells comprising the immune response cellpopulations. Agents that stimulate immune responses directly includecytokines, the “danger signals” (DAMPS, PAMPs, TLR agonists etc.),agonist antibodies or ligands (e.g. anti-4-1BB, CD40L, B7-1, and manyothers), inhibitors of immunosuppressive signals (antagonists of PD-1,PD-L1, CTLA4, Lag-3, TIM-3, IDO1, adenosine receptor, TGFbeta, and manyothers)

The targeting method utilizes a scFv antibody (or fragment thereof),alternatively, the targeting method is a secreted heterodimer TCRalpha/beta or gamma/delta chain. In both targeting methods, thepro-immune response agent includes IL-2, IFNalpha, IL-12, IL-15, IL-21,any TLR agonist and any immune checkpoint antibody (or fragmentthereof).

Example 5: The Targeting Method Utilizes a Single Chain VariableFragment (scFv) Antibody (or Fragment Thereof), or Secreted HeterodimerTCR Alpha/Beta or Gamma/Delta Chains Fused to Cell Recruiting Moieties

Another method for recruitment of immune effector cells is thedevelopment of bipecific T-cell Engagers (e.g. BiTEs) which consist oftwo scFvs connected by a linker One arm is an anti-TAA scFv, and thesecond scFv binds to the CD3ε subunit of the TCR complex and thereforetriggers T cell activation. Importantly, BiTEs and related molecules(DARTS, diabodies, fCabs etc) elicit repeated rounds of tumor cell lysisby T cells at very low effector/target (E/T) cell ratios. Thecytotoxicity is mediated by membrane perforation and subsequentinduction of granzymes and apoptosis, exactly the type of killing thatone wished to elicit in the anti-tumor setting. Normally, the affinityof the anti-TAA scFv tumor-associated antigen is much higher than thatdirected against CD3—this enforces specificity for the targeted tumor.Many BiTEs have been generated, including BiTEs that target CD19, EpCAM,HER2, carcinoembryonic antigen (CEA), ephfin A2 (EphA2), CD33, andmelanoma-associated chondroitin sulfate proteoglycan (MCSP). Other BiTEsthat are in clinical studies are composed of EpCAM, prostate-specificmembrane antigen (PSMA), or CEA-binding molecules combined withCD3-binding modules.

In some examples, the targeting method utilizes an scFv antibody (orfragment thereof), alternatively, the targeting method includes asecreted heterodimer TCR alpha/beta or gamma/delta chain. In bothtargeting methods, the cell recruiting moiety is fused to anti-CD16. Inalternative examples, the cell recruiting moiety is fused to an anti-CD3moiety, and utilizes the BiTE® technology described herein.

Example 6: Transfection of a Tumor In Vivo Using an HPV Pseudovirus withan ScFv-CD19 Fusion Protein in which the ScFv Binds the TAA Her2, andthe Tumor is Killed by CD19-Directed CAR T Cells

HPV pseudovirus comprising an ScFv-CD19 fusion protein is injected IV totransduce tumor cells in a xenograft mouse model of breast cancerexpressing Her2. The transduced tumors secrete the ScFv-CD19 fusionprotein, resulting in coating of tumor cells with the ScFv-Cd19 fusionprotein via ScFv binding to Her2. Addition of CD19-directed CART cellskills the tumor cells coated with CD19. CAR T cells directed to CD19 aremade by standard methods.

Example 7: Transfection of a Tumor In Vivo Using an AAV-Phage ChimericVirus with an ScFv-CD19 Fusion Protein in which the ScFv Binds the TAAHer2, and the Tumor is Killed by CD19-Directed CAR T Cells

A chimeric AAV/phage virus is injected IV to transduce tumor cells in axenograft mouse model of breast cancer expressing Her2. The transducedtumors secrete the ScFv-CD19 fusion protein, resulting in coating oftumor cells with the ScFv-CD19 fusion protein via ScFv binding to Her2.Addition of CD19-directed CART cells kills the tumor cells coated withCD19. The generation of CD19 directed CART cells is well established.

Example 8: Transfection of a Tumor In Vivo Using an HPV Pseudovirus withan ScFv-CD30 Fusion Protein in which the ScFv Binds the TAA Her2, andthe Tumor is Killed by a CD30-Targeted ADC

HPV pseudovirus comprising an ScFv-CD30 fusion protein is injected IV totransduce tumor cells in a xenograft mouse model of breast cancerexpressing Her2. The transduced tumors secrete the ScFv-CD30 fusionprotein, resulting in coating of tumor cells with the ScFv-CD30 fusionprotein via ScFv binding to Her2. Addition of CD30-directedantibody-drug conjugate (ADC) kills the tumor cells coated with CD30.Adcetris is a commercially available ADC from Seattle Genetics.

Example 9: Transduction of Cells with AAV Viral Particles EncodingFusion Proteins Results in Secreted Functional Fusion Proteins Capableof Directing CAR19 T Cell Targeting and Activation

The present Example demonstrates expression of fusion proteins fromcells transduced with AAV viral particles encoding fusion proteins.Further, the present example demonstrates that the expressed fusionproteins are capable of being bound by the anti-CD19 antibody FMC63 anddetected by an anti-His antibody binding to the C-terminal His tag. Onceexpressed the fusion proteins are able to activate CAR19 T cells in thepresence of cells that are CD19 negative.

The following table lists the various constructs tested in this example:

Amino Nucleotide Acid SEQ SEQ Construct # Description ID NO: ID NO: 1CD19-D1 + 2-Trastuzumab scFv 1 2 (VH/VL) 3 CD19 D1 + D2-MOC31 scFv 3 4(VH/VL) 5 CD19 D1 + D2-LY2875358 scFv 5 6 (VH/VL) 7 CD19 D1 +D2-Panitumumab 7 8 scFv (VH/VL)Methods

A 12 well plate was seeded with either A431 or 293T cells around 4×10e5cells/well in DMEM+10% FBS media. The following day, one well wascounted to get an accurate count for the viral infections. Cells wereinfected with a multiplicity of infection (MOI) of 1×10⁶ or 5×10⁶. AAV2viral particles were made by Vigene (Rockville, Md.). The viralparticles were generated from plasmids where the inserts containing theCD19 D1+D2-scFv-His sequences were cloned into pAV-FH. The viralparticles AAV-1 (CD19-D1+2-Trastuzumab scFv (VH/VL), SEQ ID NO:1), AAV-3(CD19 D1+D2-MOC31 scFv (VH/VL), SEQ ID NO:3), AAV-5 (CD19D1+D2-LY2875358 scFv (VH/VL), SEQ ID NO:5) and AAV-7 (CD19D1+D2-Panitumumab scFv (VH/VL), SEQ ID NO:7) had titers of 8.39×10¹³,1.51×10¹⁴, 3.03×10¹⁴ and 2.13×10¹⁴ GC/ml, respectively. Infections weredone in 0.6 ml/well DMEM+2% FBS where the virus was added directly intothe media. Different concentrations of virus were used from 10⁴ (aka“104” or “10e4”) to 5×10⁶ (aka “5×106” or “5×10e6”). The following day,the media was changed to DMEM+10% FBS and the incubation continued for 3or 6 days.

ELISA

Expression analysis was examined in the supernatant using an ELISA whereanti-CD19 FMC63 was used for capture and anti-His used for detection.Briefly, 96 well plates (Pierce, Cat#15041) were coated with 1.0 μg/mlreagent in 0.1 M carbonate, pH 9.5 for O/N at 4 C. The plates were thenblocked with 0.3% nonfat dry milk (NFD) in TBS (200 μl/well) for 1 hr atRT. Plates were then washed 3× with wash buffer (1× TBST: 0.1 M Tris,0.5 M NaCl, 0.05% Tween20). Titrations were performed from undilutedcell culture supernatant or purified protein at 1.0 μg/ml with serial 3×dilutions, 100 μl per well and incubate for 1 h at RT. Dilution bufferis 1% BSA in 1× TBS (0.1 M Tris, 0.5 M NaCl) followed by washing 3× withwash buffer. Secondary reagents were added (if needed) such asBiotinylated-reagents at 1 μg/ml concentration at RT for 1 hour.HRP-conjugated reagents were added at 1:2000, applied 100 μl per well,incubated at RT in dark for 1 hr. 100 μl 1-Step Ultra TMB-ELISA (ThermoFisher, Prod#34028) was added per well. Plates were read at 405 nm whencolor had developed.

XTT Cell Proliferation Assay (ATCC, Cat#30-1011K)

An aliquot of the XTT reagent and the activation reagent was rapidlythawed at 37° C. prior to use. 0.1 ml of activation reagent was thenadded to 5.0 ml of the XTT reagent. 50 μl of the activated-XTT solutionwas then added to each well. The plate was placed in the cell cultureincubator for 2-4 hours and monitored for color development. Theabsorbance of the plate was read at wavelength 450 nm. The % cell death(aka cytotoxicity) was calculated as follows:% killing=[1−OD(experimental wells-corresponding number of Tcells)/OD(tumor cells without T cell-medium)]×100Interferon Gamma Concentration Assay by ELISA

A 96 well plate (Pierce, product #15041) was coated with 1.0 μg/ml mouseanti-human IFNγ (BD Pharmingen, Cat#551221) in 0.1 M carbonate buffer,pH 9.5, overnight at 4° C. The plate was blocked with 0.3% non-fat drymilk solution in tris-buffered saline (TBS) using 200 μl/well for 1 hourat room temperature. The plate was washed ×3 with wash buffer (1×TBS/Tween: 0.1 M Tris, 0.5 M NaCl, 0.05% Tween20). 100 μl culturesupernatant from the 24 hour or 48 hour culture plates (see above) wereadded to the ELISA plate. A titration of recombinant human IFNγ (ThermoFisher, Cat# RIFNG100) was also performed in the same plate from 300ng/ml with serial 3× dilutions to 2 pg/ml to generate a standard curve.The plate was then incubated for 1 hour at room temperature. Thedilution buffer was 1× TBS (0.1 M Tris, 0.5 M NaCl) plus 1% BSA. Theplate was washed ×3 with wash buffer. Biotinylated mouse anti-human IFNγ(BD Pharmingen, Cat#554550) was added at 1 μg/ml concentration and theplate was incubated at room temperature for 1 hour. The plate was washedagain ×3 with wash buffer. HRP-conjugated Streptavidin (Thermo Fisher,Cat#21130) was added at a 1:2000 dilution from the stock, with 100 μladded per well. The plate was then incubated at room temperature for 1hour in the dark. The plate was washed again ×3 with wash buffer. 100 μlper well of 1-Step Ultra TMB-ELISA development solution (Thermo Fisher,Cat #34028) was added per well. The plate was read at wavelength 405 nmwhen color developed sufficiently.

Results

FIGS. 2A-2C demonstrate expression of fusion proteins from transducedcells. FIG. 2A shows detection of fusion protein CD19-D1+2-TrastuzumabscFv (VH/VL) (AAV#1) is expressed after transduction of A431 or 293Tcells with AAV viral particles encoding the fusion protein. Theexpressed fusion protein is capable of being bound by the anti-CD19antibody FMC63 and detected by an anti-His antibody binding to theC-terminal His tag. FIG. 2B demonstrates detection of expression offusion proteins AAV-3 (CD19 D1+D2-MOC31 scFv (VH/VL), SEQ ID NO:3),AAV-5 (CD19 D1+D2-LY2875358 scFv (VH/VL), SEQ ID NO:5) and AAV-7 (CD19D1+D2-Panitumumab scFv (VH/VL), SEQ ID NO:7) resulting from transductionof 293T cells with AAV particles encoding the indicated fusion protein.FIG. 2C demonstrates detection of expression of fusion protein AAV-3(CD19 D1+D2-MOC31 scFv (VH/VL) resulting from transduction of A431 cellswith AAV particles encoding the fusion protein.

FIGS. 3A and 3B show a summary of results of the IFNγ ELISA measuringinduction of IFNγ upon incubation of CAR19 T cells (Promab) withAAV-1-expressed supernatant and BT474 cells. FIG. 3A shows the resultsof the IFNγ ELISA at 24 hrs at a 10:1 effector:target ratio. FIG. 3Bshows the results of the IFNγ ELISA at 48 hrs at a 10:1 effector:targetratio.

FIGS. 4A-4C show induction of cytotoxicity upon incubation of CAR19 Tcells (Promab) with AAV-1-expressed supernatant and BT474 cells. FIG. 4Ashows summary XTT-cytotoxicity results for 2:1 effector:target ratioafter 24 hours. FIG. 4B shows summary XTT-cytotoxicity results for 2:1effector:target ratio after 48 hours. FIG. 4C shows summaryXTT-cytotoxicity results for 10:1 effector:target ratio after 48 hours.These results demonstrate that AAV transduction of cells (e.g. tumorcells) can result in expression of functional fusion proteins which candirect CAR19 T cell activation and cytotoxicity.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

Listing of Sequences

SEQ ID NO. 1 MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK VEIKRTSRHHHHHHSEQ ID NO. 2 ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGGGGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGGTGGAGGTGGGTCTGAGGTGCAGCTGGTGGAGTCTGGTGGTGGTCTTGTTCAACCTGGTGGTTCTCTTCGTCTTTCTTGTGCTGCTTCTGGTTTTAATATTAAAGATACTTATATTCATTGGGTTCGTCAAGCTCCTGGTAAAGGTCTTGAATGGGTTGCTCGTATTTATCCTACTAATGGTTATACTCGTTATGCTGATTCTGTTAAAGGTCGTTTTACTATTTCTGCTGATACTTCTAAAAATACTGCTTATCTTCAAATGAACTCTCTTCGTGCTGAAGATACTGCTGTTTATTATTGTTCTCGTTGGGGTGGTGATGGTTTTTATGCTATGGATTATTGGGGTCAAGGTACTCTTGTCACCGTCTCCTCAGCTAGCACCGGGGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGACATCCAGATGACCCAGTCTCCTTCTTCTCTTTCTGCTTCTGTTGGTGATCGTGTTACTATTACTTGTCGTGCTTCTCAAGATGTTAATACTGCTGTTGCTTGGTATCAACAAAAACCTGGTAAAGCTCCTAAACTTCTTATTTATTCTGCTTCTTTTCTTTATTCTGGTGTTCCTTCTCGTTTTTCTGGTTCTCGTTCTGGTACTGATTTTACTCTTACTATTTCTTCTCTTCAACCTGAAGATTTTGCTACTTATTATTGTCAACAACATTATACTACTCCTCCTACTTTTGGTCAAGGTACCAAGGTGGAGATCAAACGTACGTCTAGACATCATCACCATCACCAT SEQ ID NO. 3MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEDKKPGESVKISCKASGYTFTNYGMNWVRQAPGQGLKWMGWINTYTGESTYADDPKGRFAFSLDTSASTAYLQLSSLRGEDTAVYFCARFAIKGDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSDIVMTQSPLSLEVSPGEPASISCRSTKSLLHSDGITYLYQYLQKPGQSPQLLIYQLSNLASGVPDRFSSSGSGTDFTLKISRVEAEDEGTYYCAQNLEIPRT FGQGTKLEIKRTHHHHHHSEQ ID NO. 4 ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGGAGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGGAGGAGGTGGGTCTCAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGACAAGAAGCCCGGCGAGAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACCTTCACCAACTACGGCATGAACTGGGTGAGGCAGGCCCCCGGCCAGGGCCTGAAGTGGATGGGCTGGATCAACACCTACACCGGCGAGAGCACCTACGCCGACGACTTCAAGGGCAGGTTCGCCTTCAGCCTGGACACCAGCGCCAGCACCGCCTACCTGCAGCTGAGCAGCCTGAGGGGCGAGGACACCGCCGTGTACTTCTGCGCCAGGTTCGCCATCAAGGGCGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGCCAGCACCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGTGATGACCCAGAGCCCCCTGAGCCTGGAGGTGAGCCCCGGCGAGCCCGCCAGCATCAGCTGCAGGAGCACCAAGAGCCTGCTGCACAGCGACGGCATCACCTACCTGTACTGGTACCTGCAGAAGCCCGGCCAGAGCCCCCAGCTGCTGATCTACCAGCTGAGCAACCTGGCCAGCGGCGTGCCCGACAGGTTCAGCAGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACGAGGGCACCTACTACTGCGCCCAGAACCTGGAGATCCCCAGGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCCATCATCACCATC ACCAT SEQ ID NO. 5MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPGGGGSGGGGSGGGGSGGGGSQVQLVQSGAEDVKPDASVKLSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRRGTTYNQKFEGRVTMTTDTSTSTAYMQLSSLRGEDTAVYYCARNAWLDYWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLEASVGDRVTITCSVSSSVSSIYLHWYQQKPGKSPKLLIYSTSNLASGVPDRFSGSGSGTDFTLTISSLQAEDEGTYYCQVYSGYPLTFGGGT KLEIKRTHHHHHHSEQ ID NO. 6 ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGGAGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGGAGGAGGTGGGTCTCAGGTTCAGCTGGTGCAGTCTGGTGCTGAGGATGTGAAGCCTGATGCCTCAGTGAAGCTCTCCTGCAAGGCTTCTGGTTACACATTCACTGACTACTACATGCACTGGGTGCGTCAGGCCCCTGGTCAAGGTCTTGAGTGGATGGGTCGTGTTAATCCTAACCGGAGGGGTACTACCTACAACCAGAAATTCGAGGGCCGTGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGCAGCTGAGTAGCCTGCGTGGTGAAGACACGGCCGTGTATTACTGTGCGCGTGCGAACTGGCTTGACTACTGGGGCCAGGGCACCACCGTCACCGTCTCCTCCGCCTCCACCGGGGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGGTGGAGGTGGGTCTGACATCCAGATGACCCAGTCTCCATCCTCCCTGGAGGCATCTGTAGGAGACAGAGTCACCATCACTTGCAGTGTCAGCTCAAGTGTATCCTCCATTTACTTGCACTGGTATCAGCAGAAACCAGGGAAAAGCCCTAAGCTCCTGATCTATAGCACATCCAACTTGGCTTCTGGAGTCCCAGATAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAAGCCGAAGATGAGGGCACTTACTACTGTCAAGTCTACAGTGGTTACCCGCTCACGTTCGGCGGAGGGACCAAGCTGGAGATCAAACGAACTCATCATCACCAT CACCAT SEQ ID NO. 7MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPGGGGSGGGGSGGGGSGGGGSQVQLQESGPGDVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTTFSLQLSSVTGEDTAIYYCVRDRVTGAFDIWGQGTTVTVSSASTGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLEASVGDRVTITCQASQDISNYLNWYQQKPGKSPKLLIYDASNLETGVPDRFSGSGSGTDFTFTISSLQAEDEGTYFCQHFDHLPLAFGGGTKLEIKRTH HHHHH SEQ ID NO. 8ATGCCACCTCCTCGCCTCCTCTTCTTCCTCCTCTTCCTCACCCCCATGGAAGTCAGGCCCGAGGAACCTCTAGTGGTGAAGGTGGAAGAGGGAGATAACGCTGTGCTGCAGTGCCTCAAGGGGACCTCAGATGGCCCCACTCAGCAGCTGACCTGGTCTCGGGAGTCCCCGCTTAAACCCTTCTTAAAACTCAGCCTGGGGCTGCCAGGCCTGGGAATCCACATGAGGCCCCTGGCCATCTGGCTTTTCATCTTCAACGTCTCTCAACAGATGGGGGGCTTCTACCTGTGCCAGCCGGGGCCCCCCTCTGAGAAGGCCTGGCAGCCTGGCTGGACAGTCAATGTGGAGGGCAGCGGGGAGCTGTTCCGGTGGAATGTTTCGGACCTAGGTGGCCTGGGCTGTGGCCTGAAGAACAGGTCCTCAGAGGGCCCCAGCTCCCCTTCCGGGAAGCTCATGAGCCCCAAGCTGTATGTGTGGGCCAAAGACCGCCCTGAGATCTGGGAGGGAGAGCCTCCGTGTCTCCCACCGAGGGACAGCCTGAACCAGAGCCTCAGCCAGGACCTCACCATGGCCCCTGGCTCCACACTCTGGCTGTCCTGTGGGGTACCCCCTGACTCTGTGTCCAGGGGCCCCCTCTCCTGGACCCATGTGCACCCCAAGGGGCCTAAGTCATTGCTGAGCCTAGAGCTGAAGGACGATCGCCCGGCCAGAGATATGTGGGTAATGGAGACGGGTCTGTTGTTGCCCCGGGCCACAGCTCAAGACGCTGGAAAGTATTATTGTCACCGTGGCAACCTGACCATGTCATTCCACCTGGAGATCACTGCTCGGCCAGGAGGAGGTGGGTCTGGAGGTGGAGGATCTGGTGGAGGTGGGTCTGGAGGAGGTGGGTCTCAGGTGCAGCTGCAGGAGAGCGGCCCCGGCGACGTGAAGCCCAGCGAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCGTGAGCAGCGGCGACTACTACTGGACCTGGATCAGGCAGAGCCCCGGCAAGGGCCTGGAGTGGATCGGCCACATCTACTACAGCGGCAACACCAACTACAACCCCAGCCTGAAGAGCAGGCTGACCATCAGCATCGACACCAGCAAGACCACCTTCAGCCTGCAGCTGAGCAGCGTGACCGGCGAGGACACCGCCATCTACTACTGCGTGAGGGACAGGGTGACCGGCGCCTTCGACATCTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGCCAGCACCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGGAGGCCAGCGTGGGCGACAGGGTGACCATCACCTGCCAGGCCAGCCAGGACATCAGCAACTACCTGAACTGGTACCAGCAGAAGCCCGGCAAGAGCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGAGACCGGCGTGCCCGACAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGGCCGAGGACGAGGGCACCTACTTCTGCCAGCACTTCGACCACCTGCCCCTGGCCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCCAT CATCACCATCACCAT

What is claimed is:
 1. An adeno-associated viral (AAV) vector, or achimeric AAV/phage (AAVP) vector, comprising a nucleotide sequenceencoding a secreted fusion protein comprising: (a) an antibody, orantigen-binding fragment thereof, that binds a tumor antigen; and (b) apolypeptide antigen wherein the polypeptide antigen: (i) is not a targetfor endogenous immune cells in an individual; and (ii) is a targetantigen for an administered therapeutic selected from the groupconsisting of: cellular therapeutics, antibodies, or antibody-drugconjugates.
 2. The vector of claim 1, wherein the polypeptide antigen isa B cell antigen.
 3. The vector of claim 2, wherein the B cell antigenis CD19 or CD22.
 4. The vector of claim 1, wherein the tumor antigen isHER-2/neu, c-met, EGFR, Ga733\EpCAM, CD20, ROR1, or BCMA.
 5. The vectorof claim 1, wherein the antigen-binding fragment is an Fab, scFv, Fv, orVHH.
 6. The vector of claim 1, wherein the cellular therapeutic is aCAR-T cell or CAR-NK cell.
 7. A method of treating a subject having atumor, comprising administering to the subject: (i) an adeno-associatedviral (AAV) vector, or a chimeric AAV/phage (AAVP), vector comprising anucleotide sequence encoding a fusion protein comprising: (a) anantibody, or antigen-binding fragment thereof, that binds a tumorantigen; and (b) a polypeptide antigen wherein the polypeptide antigen:(1) is not a target antigen for endogenous immune cells in anindividual; and (2) is a target antigen for an administered therapeuticselected from the group consisting of: cellular therapeutics,antibodies, or antibody-drug conjugates; and (ii) the cellulartherapeutic, antibody, or antibody-drug conjugate, wherein the cellulartherapeutic, antibody, or antibody-drug conjugate binds to the fusionprotein, and said binding induces killing of the tumor, thereby treatingthe subject.
 8. The method of claim 7, wherein the polypeptide antigenis a B cell antigen.
 9. The method of claim 8, wherein the B cellantigen is CD19 or CD22.
 10. The method of claim 7, wherein the tumorantigen is HER-2/neu, c-met, EGFR, Ga733\EpCAM, CD20, ROR1, or BCMA. 11.The method of claim 7, wherein the antigen-binding fragment is an Fab,scFv, Fv, or VHH.
 12. The method of claim 7, wherein the AAV vector orAAVP vector transforms a tumor cell in the subject.
 13. The method ofclaim 12, wherein the transformed tumor cell secretes the fusionprotein.
 14. The method of claim 13, wherein the fusion protein binds atumor antigen on the transformed tumor cell.
 15. The method of claim 13,wherein the fusion protein binds a tumor antigen on a non-transformedtumor cell.
 16. The method of claim 14 or claim 15, wherein the cellulartherapeutic is a CAR-T cell or CAR-NK cell.